Comprehending the relationship between the body's proteins and how they change is essential to understand disease. With the completion of the human genome, defining the functional role of proteins and protein interactions is the next stage of proteomics and structural biology. Most cellular processes are performed and regulated by proteins acting in macromolecular complexes. New technologies based on mass spectrometry (MS) will be developed to facilitate the characterization of the proteome and its protein assemblies. The application of electrospray ionization mass spectrometry (ESI-MS) for studying noncovalent complexes has utility in biomedical research, and is a powerful means for investigating protein molecular recognition. Measuring molecular mass provides structural information on macromolecular assemblies, as it provides a direct determination of the stoichiometry of the binding partners in the complex. Mass spectrometry and gas phase electrophoretic mobility molecular analysis (GEMMA) will be used to advance the characterization of large macromolecular machines with greater sensitivity than currently practiced. The specific aims of this proposal are: (1) Develop ESI-MS methods with improved sensitivity for measuring large protein complexes (0.5-15 MDa). Collisional cooling and a microfabricated microchip ESI source will increase the sensitivity and reproducibility of protein complex analysis. (2) Develop direct ESI-MS methods to obtain structurally-relevant information on protein complexes and the energetics of the interactions. Develop crosslinking methods, affinity purification, and GEMMA-based techniques for isolating large protein complexes. (3) Apply ESI-MS and ESI-GEMMA to characterize protein complexes, such as the proteasome, the large protease that is responsible for protein degradation, proteins involved in Parkinson's disease (PD), and the ribonucleoprotein vault, a 14 MDa particle implicated in multidrug resistance.